Polo-like kinase 1 (Plk1) is one of the most attractive targets for anti-cancer therapy. Efforts to generate Plk1-specific inhibitors by targeting the catalytic activity of Plk1 have proven to be difficult due to similarities with the catalytic domains of other structurally related kinases. Here, we propose to develop a new class of mono-specific Plk1 inhibitors by employing a novel approach of targeting the non-catalytic, but functionally essential, PBD of Plk1. To this end, we have carried out a high throughput screen in collaboration with National Center for Advancing Translational Sciences (NCATS), Bethesda, MD. My NIH X01 grant proposal was approved for this particular project. From this screen, we have identified 3,000 compounds from a primary screen, which were narrowed down to the final 2 compounds (1S and B7) through secondary medium throughput and tertiary cell-based assays. For optimization of the final two compounds, we carried out molecular modeling/docking of the parent compounds in the Plk1 PBD to design and synthesize more water-soluble analogs (in collaboration with Dr. Terry Burke at NCI-Frederick). As a result of our initial effort to optimize the 1S compound, we obtained six significantly improved compounds, which showed Plk1 PBD inhibition activity at levels similar to that of PLHSpT (Kd = 450 nM) in an in vitro ELISA. Since the original HTS leads may belong to a class known as pan-assay interference compounds, or PAINS, we will further incorporate many drug-like characteristics during the hit-to-lead optimization in order to obtain a high-impact chemical probe that ultimately exhibits the desired efficacy against the Plk1 PBD in proof-of-concept mouse tumor models. As the first step of drug discovery, we generated the co-crystal structure of Plk1 PBD with one of the lead compounds. This structural model will be used to carry out structure-activity relationship studies and further optimization of the compounds.